In our last report we described the potentiation of CC14-induced damage by calcium in cultured hepatocytes. Potentiation of cell injury was only observed at levels of CC14 which caused moderate cell damage, indicating that calcium uptake across the plasma membrane may not be obligatory for the development of cell injury. We have therefore clairifed the role of external calcium in the development of xenobiotic induced cell injury by utilizing three compounds with different privary modes of toxicity; i.e., acetaminopen, 2,4-dinitrophenol and ionophore A23187. Addition of 100 MuM EGTA to culture medium without added calcium completely removes free calcium. Incubation of cultured hepatocytes from adult rats induced with phenobarbital in vivo in this calcium free medium with 8 mM acetaminophen resulted in extensive cell damage. The damage was greater at pH 7.6 than at pH 7.2. Addition of physiological concentrations of calcium after 2 hr incubation resulted in a dramatic decrease in apparent toxicity. These changes appeared to be unrelated to alterations in acetaminophen metabolism but rather to changes in sensitivity to cell damage.